Expression And Biological Activity Analysis Of Intrcellular ScFv Against Human Cyclin D1 In Tumor Cells

Cyclin D1, a putative mammalian G1 cyclin, plays an important role in controlling the transition from G1 to S in cell cycle. Amplification and over-expressi- on of Cyclin D1 has been found in tumorigenesis of several types of human cancer. It becomes a potential target for the treatment of human diseases in which the control of cell proliferation is deregulated.At present, there are several other methods to inhibit biological activity of molecules intracellularly. RNA based therapies, such as ribozymes or antisense RNA or RNAi are less stable and can only be used against RNA. Other protein-based therapies, such as dominant negative inhibitors, rely on competition with a native binding species and therefore require excessive concentrations In addition, these targets generally are proteins. Whereas intrabodies, by linking appropriate intracellular trafficking signal sequences to their coding genes, can be directed to all intracellular compartments including protein, nucleic acid, carbohydrate, lipid, or other cellular components. Recently intrabodies have been studied in terms of treatments for tumor growth and even clinical trials for cancer. As potential direct therapeutics, intrabodies are distinguished from small-molecule and peptide drugs in their enhanced targetbinding specificity and stability. Thus intrabody technique may pioneer a new approach for cancer therapy.In present study, an expression plasmid pER-ADic habouring an endoplasmic reticulum(ER)-retained ER-ADk scFv gene against human Cyclin Dl was constructed. In order to test the expression and activity of pER-ADic, MCF-7 cells and HeLa cells were transfected with pER-ADK and pcDNA3.1, respectively. Then expression and subcellular distribution of ER-ADk was detected by RT-PCR, Dot blot and fluorescence staining analysis. MTT analysis was used for cell proliferation analysis, the cell cycle and apoptosis changes of the transfection cells were detected by Flow Cytometry.Confocal microscope analysis demonstrated that both MCF-7/pER-ADK and HeLa/pER-ADK cells showed positive staining in cytoplasm, but the MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells did not show any fluorescence staining. RT-PCR analysis demonstrated that the ER-ADk mRNA was expressed only in MCF-7/pER-ADK and HeLa/pER-ADK cells, not in MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells. Dot blotting analysis showed that ER-ADk was detected only in MCF-7/pER-ADK and HeLa/pER-ADK cells, but was not detected in MCF-7, MCF-7/pcDNA3.1, HeLa, and HeLa/pcDNA3.1 cells. These results suggested that ER-ADk gene was already transferred into MCF-7 and HeLa cells and was successfully expressed.The growth curve showed that the expression of ER-ADranhibited the growth of MCF-7 cells and HeLa cells significantly. Cell cycle analysis by FCM showed that the expression of ER-ADk arrested the cell cycle of MCF-7/pER-ADK and HeLa/pER-ADK cells at the Gl phase, inhibiting the cells to enter the S phase of MCF-7/pER-ADK and HeLa/pER-ADK cells. Cell apoptosis analysis indicated that expression of ER-ADk inducedMCF-7/pER-ADK and HeLa/pER-ADK cells apoptosis distinctly.In conclusion, stable expression of ER-ADk regulate cell cycle and cell proliferation of MCF-7/pER-ADK and HeLa/pER-ADK cells. These data strongly suggested that intracellular scFv may become an effective approach for tumor gene therapy.